RU2027755C1 - Strain of bacterium corynebacterium krestovnikova-troitskaya used for immunostimulator preparing - Google Patents

Abstract

FIELD: medicinal microbiology. SUBSTANCE: strain of bacterium Corynebacterium Krestovnikova-Troitskaya GISK number 227 is obtained which is used for preparing of immunostimulator. Strain is nonreactive, shows low allergicity, stimulates nonspecific resistance and immunoreactivity of organism. Strain is used for preparing of vaccines showing immunogenic properties against different forms of neoplasms, leucosis, mammary, ovary and stomach cancer. EFFECT: enhanced effectiveness of immunostimulator.

Description

 The invention relates to medical microbiology and can be used to obtain a vaccine having immunogenic properties against various forms of neoplasms: leukemia, breast cancer, ovary, stomach, etc.

 Currently, unidentified strains of the genus Corynebacterium in the form of an auto vaccine are used for the treatment of cancer patients [1 and 2].

 For the treatment of patients with neoplasms of the bladder, a BCG strain with immunogenic properties against tuberculosis is used. The strain was isolated from an animal with tuberculosis and represents gram-positive bacilli, characterized by a slow, 18-20 days, growth on special nutrient media [3].

 BCG strain (Mycobacterium bovis Calmette and Guerin), being a vaccine, is used as a live vaccine to create specific immunity against tuberculosis. The strain has the ability to increase the resistance of the microorganism by activating general immunological reactions along with a specific effect, expressed in the development of post-vaccination specific immunity against tuberculosis.

A disadvantage of BCG vaccine produced from said strain, in the treatment of cancer patients is the administration of large doses and reactogenicity as a sharp increase in body temperature to 39-40 ° ^C under the first two to three injections of the drug, as well as the ability to elicit an allergy organism.

 The aim of the invention is to obtain a strain of Corynebacterium Krestovnikova-Troitscaya N277 (Corynebacterium CT-227) non-reactogenic, with low allergenicity, stimulating non-specific resistance and immunoreactivity of the body, effective in small doses of non-specific therapy of patients with various forms of neoplasms by increasing the general non-specific specific reactivity and immune reactions, the genus of the strain Corynebacterium, species Krestovnikova-Troitskaya N227.

 This goal is achieved by the fact that for vaccine therapy of patients with neoplasms, a strain of gram (+) bacteria (Corynebacterium CT-227) Corynebacterium Krestovnikova-Troitskaya N227, obtained from the blood of a cancer patient Cr.Mammae, was proposed.

 The strain of corynebacteria KT-227 is stored in the collection of the State Research Institute for Standardization and Control of Medical and Biological Preparations named after L.A. Tarasevich at number 227.

 Cultural and morphological characters.

 Rod-shaped cells, motionless, capsuleless, gram-positive, on dense nutrient media form dotted round transparent colonies with a diameter of 0.5 mm. Gram stain is positive.

The strain after rehydration grows under aerobic conditions on nutrient agar pH 7.6-7.8 with the addition of 0.2% glucose and 10% inactivated horse serum or Tween 80 (0.05%). The growth temperature is 37 ° ^C. The duration of 24-48 hours growth strain possesses pigment cream on media with serum, yellow -. In environments with Tween.

 Biochemical properties.

 The strain does not utilize beckons, galactose, sucrose, lactose, raffinose, inulin, remnose, dulcite, adonite, xylose, rabose, arabinose, sorbitol, starch. It utilizes glucose and maltose before acid formation. It has the enzyme catalase.

 It does not have enzymes: gelatinase, urease, oxidase, does not hemolize red blood cells, does not produce indole and hydrogen sulfide. Does not reduce nitrates.

 The sensitivity of the strain to antibiotics was studied by the disk diffusion method. The strain is sensitive to oxylline, ampicillin, ristomycin, polymexin, benzylpenicillin, carbenicillin, methicillin, erythromycin, tetracycline, oleandomycin, gentamicin, kanamycin, neomycin, monomycin, streptomycin [4 and 5].

 The strain is stored in ampoules, it remains viable as a result of freeze drying from a frozen state in a protective sugar gelatin medium.

 The strain has an oncolytic activity equal to 98 ± 2% [6]. (A sarcoma strain was used as a tumor model).

PRI me R. To obtain a strain of corynebacteria KT-227 take nutrient agar pH 7.6-7.8 with 0.2% glucose, 10% inactivated horse serum or 0.05% Tween 80, incubation at 37 ^about 24-48 hours.

 To obtain a vaccine from a strain, the culture is grown on a special nutrient medium. The resulting biomass is washed off with a sterile solution, packaged in ampoules and sealed.

 To confirm the possibility of using them as vaccines, the most typical cultures with a certain level of immunostimulating effect revealed in animal experiments were selected from among the studied cultures. They were subsequently used as a vaccine for the preparation of an immunostimulant.

 The modulating effect of the drug "in vivo" on mice was studied. As a result of the studies, it was shown that the drug has an immunostimulating effect against IgY antibody formation. The greatest immunostimulating effect was found when using the drug on the day of immunization. The data obtained indicate a pronounced immunostimulating effect of the drug. Moreover, this effect is manifested in relation to the inductive phase of the immune response.

 In experiments on mice of different lines with spontaneously developing tumors, as well as infected with a tumor suspension of the S-180 sarcoma strain and on the model of transplantable hemocytoblastosis, it was shown that the use of the vaccine from the strain in the first case led to an increase in the incubation period or to prevent the development of spontaneous leukemia and increase life expectancy sick animals. In animals infected with a tumor strain, the use of a vaccine helped to restore immunological non-specific resistance and reduce the percentage of diseased animals. In connection with the restoration of immunological non-specific resistance, tumor growth was inhibited from 74 to 56.6%, depending on the timing and route of administration of drugs.

 For histological examination, the injection site, internal organs and organs of immunogenesis were taken. It was found that against the background of a decrease in lipid peroxidation (LP) and an increase in the number of SH groups in the tissues of the spleen and liver, activation of mechanisms inhibiting the growth of transplanted tumors took place.

 When assessing harmlessness in experiments on laboratory animals, the acute and chronic toxicity of the vaccine strain was verified. Both the strains and the vaccine 24 hours after intravenous administration in experiments on the study of acute toxic effects caused small focal reactive changes in the pulmonary parenchyma, which reduced to moderate edema of the interalveolar septa with their mainly lymphoid and microphage infiltration. The indicated reaction disappeared and was not recorded 7 days after intravenous administration of these reagents. No abnormalities were found in other internal organs (central nervous system, heart, liver, kidneys, pancreas, adrenal glands, or intestines). No pathological reactions were noted in the organs of the immune system.

 With a single intramuscular injection of the strain and vaccine in experiments on acute toxicity, changes were detected 24 hours after injection at the injection site, where focal leukocyte infiltration and destruction of individual muscle elements occurred. These changes were accompanied by moderate hyperplasia of the regional lymph nodes without disturbing their structure and myelosis. In this period, reactive changes in the lung tissue with infiltration of the interalveolar septa were recorded in vaccine-treated animals. In other organs and the immune system, abnormalities were detected. After 7 days after the intramuscular injection of the strain and the vaccine, there were no signs of pathological changes both at the injection site and in the other organs studied.

 Tenfold intramuscular administration of the strain and vaccine in experiments of chronic toxicity did not lead to the development of any disturbances both at the injection site and in other organs of the immune system.

 The data obtained convincingly show the absence of the toxic effect of the doses used in experiments on the study of acute and chronic toxicity of the strain of corynebacteria KT-227 and a vaccine prepared on its basis.

 Data on the activation of an immune system that indirectly provides protection against a malignant agent.

 The strain is proposed as a vaccine (as a drug with the ability to normalize cellular and humoral immunity) for non-specific treatment of patients, in particular with various forms of neoplasms.

 Due to the fact that the prepared vaccine from the strain does not have a specific therapeutic effect on growth, but indirectly affects the normalization of the immune system (humoral and cellular) by restoring the natural resistance to the tumor process, the vaccine was designated as an immunostimulant with the name "corynostim". It is recommended for use in combination therapy with other drugs.

 When evaluating the immunostimulating effect, studies were conducted on two groups of cancer patients treated in two ways.

 The first group (control) - 27 people, the second group (treated with the drug) - 23 people. The functional activity of neutrophils and monocytes and some other characteristics of cellular immunity were determined. Gumbral immunity was determined by the level of immunoglobulins of classes I, A, M, IgE.

 It was shown that the two groups reliably differ, and the significance of the differences remains, even if the immunity indicators are divided into two groups: humoral and cellular. Relative differences were revealed in the group treated with the drug as compared to the control: a decrease in the level of IgA, percentage of T-lymphocytes, an increase in oxygen-dependent bactericidal activity under load of the drug. The cause of significant differences in immunological parameters in these two groups should be considered the use of the drug in one of them.

 It follows that this drug has an immunomodulatory effect. One of the hypothetical mechanisms of the action of this drug on the immune system is the activation of normal K cells, which becomes possible due to the almost complete elimination of suppressors.

 Data on the increase of non-specific protection factors after the introduction of the proposed strain.

Experimental studies and long-term treatment of cancer patients with autologous vaccines from this type of bacteria, as well as limited clinical trials for the treatment of 52 patients with corinestim, allow us to draw a conclusion about the effectiveness of the drug in the complex treatment of patients with cancer, which is manifested in the following:
increased cytotoxicity in relation to cells with impaired parameters is simple, accelerating the clearance and digestion of metabolites and products of destruction of tumor cells, in the disappearance of toxicosis in patients;
inhibition of tumor growth and inhibition of the process of metastasis;
enhancing the effectiveness and reducing the toxicity of anti-blastoma drugs;
reduction to a minimum or the abolition of blood transfusions or blood substitutes;
eliminating the need for prolonged use of pain-relieving pharmaceuticals;
increase the life expectancy of patients with cancer;
a sharp decrease in the frequency of secondary bacterial infections associated with opportunistic microbes.

Claims (1)

 CORYNEBACTERIUM KRESTOVNIKOVA - TROITSKAYA BACTERIA STRAIN USED FOR PREPARING THE IMMUNITY OF THE MULATOR.  The bacterial strain Corynebacterium Krestovnikova - Troitskaya GISK N 227 used to prepare the immunostimulant.